Preservation of genome integrity is essential for the health and viability of all organisms, including humans. Understandably, all organisms have multiple mechanisms of maintaining genome stability. One of these systems is the replication checkpoint, which detects stalled or broken replication forks. It protects genome integrity by arresting cell division, by stabilizing the stalled forks, and by ensuring an adequate supply of replication proteins and nucleotides needed to complete DNA synthesis once the impediments to fork progression are removed. In humans, mutations that impair the replication checkpoint are causally associated with genomic instability, developmental, neurological and immunological diseases, and a predisposition for cancer. Deciphering how this checkpoint works, and more generally what happens when replication forks stall, rearrange or collapse, is therefore a fascinating biological problem that also has broad implications for improving human health. This application proposes to use fission yeast to study how eukaryotic cells maintain genome integrity during DNA replication. This organism has played a central role in the discovery and analyses of genome stability mechanisms. The proposal has three specific aims. The first is to identify and characterize substrates of the replication checkpoint kinase Cds1. The second aim is to understand the role of a BRCT domain protein that is needed to tolerate replication stress. The third aim is to determine how cells recapture broken replication forks with a focus on the function of the structure-specific endonuclease Mus81. A variety of techniques will be employed: yeast molecular genetics, chromatin immunoprecipitation, protein interaction screens involving yeast two-hybrid and multidimensional protein identification technology, structural biology, deconvolution microscopy of fluorescently tagged protein in live cells, and genome-wide analyses of proteins localized to site of DNA damage using high density oligonucleotide microarrays. All of the proteins analyzed in this proposal have orthologs in humans whose functions are incompletely understood. Therefore, the findings from this study will serve as a paradigm for the investigation of genome stability mechanisms in humans.